Microbiology Essentials: Techniques, Staining, and Epidemiology

Posted on Mar 1, 2025 in Microbiology

Historical Perspectives

• Ignaz Semmelweis: Introduced hand washing with chlorinated lime in 1846, documenting a decrease in fever.
• Joseph Lister: Father of antisepsis, used carbolic acid for cleaning hands and instruments, decreasing infection after amputations and surgeries.

Hand Washing and Emulsification

• Soap: Anionic surfactants with two ends; emulsification allows removal of insoluble matter using water. Mechanical action of hand-washing is crucial.

The 5 I’s of Microbiology

• Inoculation: Transfer of a bacterium/sample to a medium.
• Incubation: Inoculated media is moved to a temperature-controlled location for growth.
• Isolation: Spaced-out growth allowing for the growth of specific bacterium.
• Inspection: Analyzing growth.
• Identification: Biochemical, genotyping, and immunologic testing.

Specificity and Sensitivity

• Specificity: Ability to specifically test for one pathogen.
• Sensitivity: Smallest amount of pathogen needed for a test to detect its presence.

Enumeration Techniques

• Streak Plate: Determines “what” is in the culture; most common method.
• Serial Dilution: Determines “how much” is in the culture; used with spread plating. (CFU/mL)
• CFU/mL: Colony forming units per milliliter of culture.
• Colony: Group of cells that are genetically identical (can be millions of cells stacked on each other).
• CFU/mL Formula: # of colonies / (dilution x amount plated). E.g., 85 / (10^4 x (150/1000)).
• Basics of Dilutions: With each consecutive transfer, the # of bacteria present in each dilution tube decreases. Sometimes referred to as 10-fold serial dilutions. 1000µL = 1mL.
• Spread Plate: Uses the entire plate, more sensitive and expensive than a spot plate. TNTC: >300, TFTC: <30.
• Spot Plate: Instead of spreading 1mL of a single dilution on the entire plate, 4 separate dilutions are spotted on one plate; spots 25µL aliquots of each dilution tube on a quadrant. TNTC: >25, TFTC: <5.
• CFU/mL Applications: Used in UTI diagnostics, food and water safety, and protein analysis.

Microbial Ecology and Isolation

• Microbes are everywhere.
• Microbial Ecology: Studies the relationship microorganisms have with their environment, plants, animals, and one another.
• Microbes complete the carbon and nitrogen cycles.
• Microbes promote plant growth and nutrient acquisition.
• Humans have normal flora/”human microbes.”
• 100 trillion (10^14) microbial cells in the human gut, 10x the number of human cells in the body.
• Microbial diversity can be found in the skin, urogenital tract, and respiratory tract.

Historical Figures in Microbiology

• Robert Hooke: Reported that living things were composed of little boxes/cells.
• Antoine van Leeuwenhoek: Constructed a microscope which could magnify up to 30-100x and called microorganisms animalcules.
• Francisco Redi: Disproved spontaneous generation.
• Lazzaro Spallanzani: Utilized heat to kill microorganisms within flasks containing broth. Critics argued that air was necessary for spontaneous generation (led to Pasteur’s experiment).
• Louis Pasteur: French chemist who utilized heat to kill microorganisms within a swan neck flask. Also developed sterilization procedures still utilized today in food, medicine, and research.
• Robert Koch: German physician who validated the germ theory of disease: microorganisms are the cause of many diseases. Found ways to grow bacteria outside of the host within a lab setting and developed techniques for culturing microorganisms within a lab.

Pure Cultures and Aseptic Techniques

• Pure Cultures: Aseptic techniques, methods, and techniques developed to work with microorganisms without contaminating the cultures or self-infection. There is a constant possibility of contamination by air.
• Agar: Solidifying agent obtained from seaweed, not degradable by most microorganisms, liquifies at 100°C and solidifies under 40°C.

Gram Stain Characteristics

Microscopy and Staining

• Bright Field Microscopy: Objects are dark, and the “field” is light.
• Stains: Chromophores that have acidic (-) and basic (+) charged ions.
• Direct/Simple Stain: Stains the organism, uses a basic stain.
• Negative/Indirect Stain: Stains the field or background; uses an acidic stain.
• Smears need a clean side for staining.
• Heat fixing some smears increases adherence, kills bacteria, and increases staining procedure success.

Bacterial Morphology and Staining Types

• Bacilli: Worm-like.
• Cocci: Dots.
• Spirilla: Spiral/web-shaped.
• Simple Stain: Bacteria uptake the stain, requires heat for fixing of smear and allows visual for size, shape, arrangement. Uses basic stains such as methylene blue, safranin, or violet.
• Negative Staining: Stains background, not bacteria; acidic stain, bacteria is less distorted and does not require heat fixation.

Differential Staining

• Differential Staining (3 parts):
  • Primary stain (imparts color to all cells).
  • Decolorizing agent (may/may not remove the primary stain from the cell).
  • Counterstain (provides color contrast to primary stain).
  Examples: Gram stain, acid-fast stain.

Isolation Techniques (Continued)

• Richard Petri: Invented the Petri dish.
• Streaking for isolation isolates bacteria.
• Good Streak: Separating mixed cultures into individual colonies, easy to tell if it is contaminated.
• Bad Streak: Short term, dries out quickly unless refrigerated; easily contaminated.
• Pure Culture: Colonies of a single morphological type are all that are present upon our streak plate.
• Mixed Population: Several different morphologies.
• Form, elevation, and margin are used to describe the different cell morphology.

Gram Stains (Continued)

• Gram (+) stain is purple, and Gram (-) is pink.
• Gram (+) 90% of cell wall, and Gram (-) 10% of cell wall plus outer lipopolysaccharide layer.
• Mechanisms of Gram Staining:
  • Primary stain (application of crystal violet dissolves into CV+ and Cl- ions).
  • Application of iodine (mordant that makes CV-I complexes).
  • Alcohol (decolorizing agent that washes away the outer layer of Gram (-); Gram (+) stays purple).
  • Counterstain with safranin, which gives Gram (-) a pink color.
• Common sources of Gram stain error: loop too hot during culture transfer, excessive heat, improper staining technique, power washing bacteria off.
• Problems with Gram staining: some bacteria will not Gram stain or will have variable results. Over-decolorization with acid alcohol can make Gram (+) look like Gram (-) (pink).
• Spore Stains: Endospores are formed by bacteria when essential nutrients or water are not available.
• Flagella: Propellers, common in Gram + and Gram – rods.
• Staining is important; it is an efficient way to identify microorganisms.
• Some bacteria contain mycolic acid in their membranes, which makes them impermeable to traditional staining procedures (acid-fast bacteria). They resist decolorization with acid alcohol; steam heat is used to facilitate the uptake of carbol fuchsin by the acid-fast bacteria. Methylene blue serves as a counterstain.

Microscopes

• Light Microscopes: Use light rays passing through lenses to magnify the object.
• Stereomicroscope/Dissection Scope: Designed to study entire objects in three dimensions at low magnification.
• Compound Light Microscope: Used for examining small or thinly sliced sections of objects under high magnification.
• Two sets for compound magnification: ocular and objective; illumination comes from below the specimen.
• Etiquette: Use the same one for the semester, carry with both hands (one under the base and the other on the arm), only clean lenses with lens paper.

Microscope Components

• Viewing Head: Holds ocular lenses.
• Arm: Supports upper parts of the scope and is used to carry it.
• Nosepiece: Revolving piece that contains the objectives.
• Objectives: Scanning (4x), low power objective (10x), high power objective (40x), oil immersion objective (100x).
• Stage: Platform that holds the slide.
• Stage Clips: Used to hold the slide in place.
• Mechanical Stage Control Knobs: Used to move the stage/object forward/back, right/left.
• Coarse Adjustment Knob: Used for initial focus with the lowest power objective.
• Fine Adjustment Knob: Used for final focus.
• Condenser: Lens system below the stage used to focus the beam of light.
• Diaphragm: Controls the amount of light reaching the specimen.
• Light Source: Attached lamp that directs light up through the object/specimen.
• Base: The flat surface the microscope rests on.
• Field of View: The circle that is visible through the ocular lenses.

Resolution and Refraction

• Resolution: Ability to see two objects separate and distinct, depends upon refraction (scattering) of light.
• Refraction can be limited by: light wavelength (shorter wavelengths = better resolution. Most scopes employ a blue filter because it has a short wavelength).
• Numerical Aperture (NA): Ability of the objective to capture scattered light, related to the curvature of the lens.

Oil Immersion Lens

• Oil Immersion Lens: Must be using the oil immersion lens, move lens to the side, add a small drop of oil, move oil immersion lens back into place, only use the fine adjustment knob, should be able to see a “seal” between the lens and slide created by the oil droplet.

Epidemiology

• Epidemiology: The study of the frequency and distribution of disease and health-related factors in human populations.
• John Snow: A physician who assisted in identifying that the London cholera outbreaks were not from bad air but from a contaminated water source. Mapped out infections and traced them back to a water pump.
• Prevalence: The total number of cases with respect to the entire population, usually represented by a percentage of a population.
• Incidence: Measures the number of new cases over a certain time period, as compared with the general healthy population.
• Incidence conveys information about the risk of contracting the disease, whereas prevalence indicates how widespread the disease is.
• Morbidity Rate: # of people afflicted with a certain disease.
• Basic Reproduction Number (R0): Number of expected infections as a result of one single case of the disease within a susceptible population (this predicts how quickly a pathogen will spread).

Stages of Clinical Infection

• Four Stages of Clinical Infection:
  • Incubation period (initial contact to first symptoms).
  • Prodromal stage (general “unwell” feeling).
  • Period of invasion (symptoms are severe).
  • Convalescent period (individual is recovering).

Types of Diseases

• Types of Disease:
  • Sporadic (occasional at irregular intervals, outbreak of shellfish).
  • Endemic (steady frequency over a period of time, Rocky Mountain fever).
  • Epidemic (prevalence is increasing more than expected, seasonal flu).
  • Pandemic (widespread, COVID-19).
• 4 Types of Carriers: Incubation carrier, convalescent carrier, chronic carrier, and passive carrier.
• Different Transmission: Droplet infection (sneeze, cough) and fomites (contaminated inanimate objects).
• Vectors: A live animal (other than a human) that transmits infectious agents from one host to another.
• Biological vectors actively participate in the pathogen life cycle in addition to transporting it; mechanical vectors only transport it.

Vector-Borne Diseases

• Vectors – Ticks: Lyme disease and Rocky Mountain Spotted Fever: Symptoms include headaches, fever, rash.
• Malaria/Yellow Fever: Parasite, spread by mosquitoes.

Communicable vs. Non-Communicable Diseases

• Communicable Disease: When an infected host can transmit the infectious agent to another host and establish infection in that host (cold, flu, STIs).
• Non-Communicable Disease: Infectious diseases do not arise through transmission from host to host.

Nosocomial Infections (HAIs)

• Nosocomial Infection or HAI: Diseases that are acquired or developed during a hospital stay.
• More than a third could be prevented; 1 in 20 patients acquire it.
• Typhoid Mary: Carried Salmonella typhi, the causative agent of typhoid fever, and spread it to many others as a cook.
• Reservoir: Location where pathogens can be found for a long period of time.

Experimental Variables

• Independent Variable: Part of the experiment that is intentionally changed.
• Dependent Variable: Can be measured; part of the experiment that will change as a result of the independent variable.
• Constant: Kept the same throughout the experiment.
• Control: Sets a baseline for reading the results of the dependent variable.
• Replicates: # of test subjects or groups that are treated the same within an experiment.

Microscope Usage

• Parfocal: In focus with one lens – in focus with all (in theory); higher magnifications may require slight adjustments.
• Microscope Use:
  1. Always begin focusing at the lowest possible point.
  2. As you switch from low to high power, the field of view becomes darker.
  3. As you switch from low to high power, the field of view becomes smaller.
• Glass and air = different refractive indexes.
• Glass and oil = almost identical refractive indexes.

Capsule Stains

• Capsule Stains: Tight arrangement is called a capsule; diffuse irregular arrangement is called a slime layer.
• Most capsules are composed of polysaccharides, are uncharged, and appear as clear halos surrounding the stained bacterium.